Photoacclimation to Long-Term Ultraviolet Radiation Exposure of Natural Sub-Antarctic Phytoplankton...

Photochemistry and Photobiology, 2006, 82: 923–935

Symposium-in-Print: UV Effects on Aquatic and Coastal Ecosystems

Photoacclimation to Long-Term Ultraviolet Radiation Exposureof Natural Sub-Antarctic Phytoplankton Communities:Fixed Surface Incubations Versus Mixed Mesocosms

Marcelo Hernando*1, Irene Schloss1,2, Suzanne Roy3 and Gustavo Ferreyra2

1CONICET, Argentina2Instituto Antartico Argentino, Cerrito 1248, C1010AAZ, Buenos Aires, Argentina3Institut des Sciences de la Mer (ISMER), Universite du Quebec a Rimouski, Rimouski, Quebec, Canada

Received 29 August 2005; accepted 24 May 2006; published online 26 May 2006 DOI: 10.1562/2005-08-29-RA-662

ABSTRACT

Solar UVB radiation (280–320 nm) is known to have detri-mental effects on marine phytoplankton. Associated with theseasonal ozone hole in Antarctica, stratospheric ozone de-pletion occasionally influences the sub-Antarctic (BeagleChannel, Argentina) region, enhancing levels of UVB. Theprimary objective of this work was to study the effects ofseveral (i.e. 6–10) days of exposure to UVB on the taxonomiccomposition and photosynthetic inhibition of local phyto-plankton communities. For different light treatments, fixed-depth incubations placed in an outdoors water tank werecompared with incubations in 1900 L mesocosms, wherevertical mixing was present. Phytoplankton growth wasinhibited by UV radiation (UVR) in fixed-depth experimentsbut not in the mixed mesocosms. Under fixed and mixedconditions alike, photosynthesis was significantly inhibited byUVB at the beginning of the experiment but no longer afterseveral days of exposure, suggesting that cells had acclimatedto radiation conditions. There was a change in species com-position in response to UVR exposure in both experiments,which likely explained acclimation. In the community exposedto fixed conditions this change was from a phytoflagellate-dominated assemblage to a community with high relativeabundance of diatoms after 6 days of exposure. UVA was re-sponsible for most of the observed growth inhibition; however,the reduction in photosynthesis was produced by UVB.The reasons behind this variability in responses to UVR areassociated with species-specific sensitivity and acclimation,and the previous light history of cells. In the community ex-posed in mesocosms, an assemblage codominated by phyto-flagellates and diatoms was observed at the beginning ofthe experiments. After 10 days of exposure, green algae(Eutreptiella sp.) had increased, and phytoflagellates were thedominant group. The synthesis of mycosporine-like aminoacids (MAAs), antioxidant enzymes and photosynthetic

antenna pigments, in relation to repair and protectionprocesses, may explain the reduced inhibition of both growthand photosynthesis that was observed in the phytoplanktoncommunity after several days of exposure. For environmentssuch as the Beagle Channel seasonally exposed to the ozonehole, the results obtained from the fixed-depth experimentsshow that species can cope with UVR by means of MAAsynthesis, while mixing would primarily promote a changein species composition and defense strategies.

INTRODUCTION

It has been shown that UVB and solar UVA radiation (320–400

nm) can have deleterious effects on phytoplankton. These radia-

tions can, among other effects, reduce cell growth rates (1), induce

DNA damage (2) and inhibit photosynthesis through effects on

photosystem II (PSII) (3) and ribulose-1,5-biphosphate carboxylase/

oxygenase (RUBISCO) (4). UVB can penetrate up to ecologically

relevant depths in the oceans (5–8). Under low ozone conditions,

UVB flux could affect primary production below 15 m in the water

column (9–11). However, Cullen and Lesser (12) and Neale et al.(13,14) suggested that the effects of UVB could be greatly

attenuated by vertical mixing. The time of residence of cells at a

certain depth (i.e. the light history of the cells [15,16]), is therefore

influenced by the mixing regime prevailing in the water column.

The degree of exposure as well as the balance between cell damage

and repair processes will then be a function of both mixing velocity

and depth (14).

UVR effects have been studied especially in Antarctic waters

due to the presence of the seasonal ozone thinning. Sub-Antarctic

waters are, however, also exposed to the so-called ozone hole,

which can periodically cover the southern tip of South America

(17). Previous studies in ocean waters around Antarctica have

shown that the tolerance to UVR varies among phytoplankton

groups (18) and that a differential tolerance exists to either

UVA or UVB (19). Phytoplankton can be protected from UVR-

induced damage by the synthesis of mycosporine-like amino acids

(MAAs), which absorb radiation in the UVR range (20–22).

Antioxidant compounds can also be generated to reduce UVR

damage (23).

*Corresponding author email: [email protected](Marcelo Hernando)

� 2006 American Society for Photobiology 0031-8655/06

923

Fixed surface incubations (i.e. not vertically mixed) conditions

represent the worst-case scenario for phytoplankton. Samples from

the natural environment are exposed to such a radiation regime

only when remaining near the ocean surface. This situation may

be possible in the area from which the samples were obtained.

Navarino and Isla Grande de Tierra del Fuego act as barriers to

give this part of the Beagle Channel the character of a semienclosed

water body. Low surface salinity (2.5–2.7 PSU (practical salinity

units) [24]) from local inputs of great amounts of glacial melt water

(24) can produce a strong stratification, which will remain as long

as intense winds are absent (25).

Vertical mixing is one of the most important variables within

the water column (26) because it exposes planktonic organisms to

fluctuating (i.e. variable) radiation regimes (27). The simulation of

mixing and of more realistic environmental conditions is only

possible when a large water mass is studied. Several UVR studies

with natural phytoplankton communities have been performed

in recent years using outdoor mesocosms (e.g. 28–30). Although

this approach is different from the natural environment, it has

advantages primarily in terms of the more natural light environ-

ment and presence of planktonic communities resembling local

conditions. Most of the earlier studies were performed on a short-

term scale (hours). However, long-term (several days) studies are

required for a better understanding of the effect of UVR radia-

tion on the phytoplankton community.

The primary goal of this work was to study the effects of several-

day exposure to UVR on the taxonomic composition, growth and

photosynthetic inhibition of sub-Antarctic phytoplankton commu-

nities. The algae were exposed to constant light environments

(photosynthetic available radiation [PAR], 400–700 nm and UVR)

under different mixing regimes, comparing surface exposure with

those of mixed, large-volume mesocosms.

MATERIALS AND METHODS

Experimental set-up. Two series of experiments were performed in Ushuaia(548529S, 688189W, Tierra del Fuego, Argentina) with surface phytoplank-ton collected from the Beagle Channel. In the first experiment (fixed-depth),performed 11–17 January 1998, phytoplankton were exposed to either UVRor PAR in 500 mL vessels. Three irradiance treatments were included:PARþUVR (NUVB treatment), PARþUVA (WUVB treatment) and PAR

only (PAR treatment). This design allows the separation of the inhibitoryeffects of UVA (WUVB) and UVB (NUVB). Cylindrical UV-transparentvessels (Plexiglas UVT, GS 2458, Rohm and Haas, Darmstadt, Germany)were used to expose phytoplankton to full solar radiation. For the WUVBtreatment, the vessels were covered with Mylar (DuPont, 50% transmissionat 323 nm), which allows cells to be exposed to UVA þ PAR. The PARtreatment was performed with cylindrical Plexiglass vessels (UF3) (Rohmand Haas), which cuts off all UVR. Three replicates were performed foreach of the treatments. The vessels were stirred manually twice a day. Thealgae were exposed to natural sunlight during 6 days. The vessels wereplaced in a water tank, with flowing thermostatic cold tap water to main-tain in situ temperature (approximately 108C). Temperature was monitoreddaily with a mercury thermometer. To prevent nutrient depletion, culturemedium (f/2 concentration [31]) was added to the different treatments atthe beginning of the experiments.

Mesocosms are defined as large outdoor enclosures (.1 m3) of water(see Belzile et al. this volume). Experiments were performed 5–15November 2001. The mesocosms (1900 L volume; Fig. 1) were made ofclear polyethylene that transmitted 85–93% of light in the 280–750 nmrange. Mesocosms were filled 1 day before the beginning of the experimentusing local seawater. Because local nutrient concentrations were low, a2 L solution with 2 lmol NO3

� and 3.3 lmol Si(OH)4 was added to themesocosms at the beginning of the study (32). Continuous mixing withinthe mesocosms was induced by water pumping (turnover rate of ca 1 h)using Little Giant pumps (model 2-MD-HC, Little Giant Pump Co.,Oklahoma City, OK). Water intake was located between 25 and 55 cmbelow the mesocosm surface, and the outflow was located at the bottomof the mesocosms. Three different light treatments (three replicates pertreatment) were used: natural ambient irradiance (NUVB), natural irra-diance plus increased UVB (HUVB) equivalent to a 60% ozone depletion(33) and without UVB irradiance (WUVB, covered with Mylar). UVBenhancement was provided by four UVB fluorescent light tubes (TL40 W/12RS, Philips, Markham, ON, Canada; Fig. 1). These tubes were preburnedfor .100 h before the experiments. Wavelengths shorter than 290 nm(UVC radiation) were screened out by cellulose diacetate film that wasreplaced daily. To ensure equal shading effects, four wooden dummy lampswere suspended over the NUVB and WUVB mesocosms. The lamps wereturned on from 1100 to 1600 h starting on day 3 of the experiment. Surfacewater temperature was continuously measured using thermocouples (type‘‘J’’) connected to a data logger (21X, Campbell Scientific, Inc., Edmonton,AB, Canada). The temperature loggers were set at 1 m and temperature wasrecorded every 10 min. Temperature was also measured at each samplingtime, with a regular mercury thermometer. Both readings agreed within0.58C, on average. Surface water salinity was determined daily usinga mini-CTD (Micro-CTD 3, Falmouth Scientific, Inc., Cataumet, MA).

Light measurements. During both experiments, incident solar radiationwas recorded for wavelengths at 305, 320, 340 and 380 nm and PAR every10 min using a GUV510 surface radiometer (Biospherical Instruments, Inc.,San Diego, CA). For the mesocosm experiment, irradiance throughout thewater column was measured every day in the center of the mesocosms witha PUV profiling UV radiometer (which records irradiances at fourwavelengths in the ultraviolet region: 305, 320, 340 and 380 nm, andPAR; Biospherical Instruments, Inc., model 500) at 1100, 1315 and 1530 h.Incident solar radiation was estimated using a multivariate model from dataobtained with an SUV-100 (Biospherical Instruments, Inc.) installed on theroof of Centro Austral de Investigaciones Cientıficas, Argentina, which islocated 4 km from the experimental site (33). The vertical attenuationcoefficient (Kd, m�1) and mean depth of 1% light penetration (Z1%, m) werealso determined for UVB (305 nm), UVA (340 nm) and PAR (400–700nm). Daily fluence at the surface was corrected for the lamp shading effect,estimated at approximately 36% (S. Dıaz, personal communication). Dailyfluence averaged over the water column was calculated using the methoddescribed by Riley (34) and is equal to the daily surface irradiance times theratio (1� e�kd * z) * (kd * z)�1 where z is the depth of the mesocosm.

Photosynthesis measurements. For both series of experiments, 50 mLsubsamples (per vessel or per mesocosm, corresponding to the fixed andwell-mixed experiments, respectively) were taken to measure primaryproduction. These subsamples were exposed to the same light treatmentsas the vessels or mesocosms from which they originated: for the naturalspectrum, phytoplankton were exposed in 50 mL cylindrical quartz tubes(NUVB treatment); for the WUVB treatment, the quartz tubes were coveredwith Mylar. PAR control experiments (PAR treatment) were performedwith cylindrical UF3 Plexiglass tubes (35). Three replicate samples wereused for each of the treatments and controls in the first set of experiments

Figure 1. Schematic illustration of the mesocosms experimental design.

924 Marcelo Hernando et al.

and one per mesocosm (six per light treatment) for the second set ofexperiments. Samples from both sets of experiments were incubated for 6 haround local noon (i.e. from 1000 to 1600 h) after inoculation of 5 lCi(0.185 MBq) of labeled (NaH14CO3) sodium bicarbonate (36). Followingincubation, the samples were filtered onto Whatman GF/F glass fiber filters(25 mm). The filters were then placed in 7 mL scintillation vials, exposed toHCl fumes overnight, dried, and counted using standard liquid scintillationtechniques (37) in a Packard Liquid Scintillation Analyzer Model 1600 TR(Canberra, Australia). For the fixed-depth experiment, 3 days were studied(i.e. days 0, 2 and 6). Mesocosm subsamples were studied daily, excepton day 4.

Algal pigment analyses and cell counts. In the fixed-depth experiments,80 mL samples were taken on days 0, 2 and 6 at 0900 h to determinechlorophyll a (chl a) concentration by fluorometry and another 20–30 mL,daily, for cell counts. Subsamples for cell counting were kept in darkbottles and fixed with formalin previously neutralized with sodium borate(final concentration 0.4%). Samples were counted with an invertedmicroscope using the Utermohl method (38). Chl a analyses (80 mLsamples for the fixed experiment, and 500 mL for the well-mixedexperiment) were extracted in 7 mL of absolute methanol (39) anddetermined by fluorometric analyses using a Turner fluorometer (model450). Chl a analyses in plankton .5 lm was performed as describedbefore, but after prefiltering the sample with Nuclepore Polycarbonatefilters (5 lm pore size). In the second series of experiments (mesocosms),algal pigments were additionally analyzed by chromatography. Thesesamples were filtered onto 25 mm GF/F filters (Whatman, Maidstone,UK), which were then frozen in liquid nitrogen and subsequently stored at�808C. Samples were sent from the experimental site to Rimouski forfurther analyses at the Institut des Sciences de la Mer de Rimouski.Methanol extracts of pigments present on the GF/F filters were analyzedby HPLC according to the method described by Zapata et al. (40).

Growth measurements. Instantaneous growth rates were determinedaccording to the following equation:

l ¼ lnðNn=Nn�1Þ ð1Þ

where l is the daily growth rate (d�1), Nn is the cell concentration on aspecific day and Nn�1 is the cell concentration on the day before.

To determine the degree of growth inhibition during the fixed ex-periments, instantaneous growth rate in phytoplankton exposed to UVR(WUVB and NUVB treatments) was compared to the average growth ofthose exposed to PAR.

UVR-absorbing compounds. UVR-absorbing compounds (MAAs) weredetermined following the technique described in Helbling et al. (41).Samples from the fixed-depth experiments were extracted for 2 h in 7 mLof methanol at 88C. After extraction, samples were centrifuged and thesupernatant was scanned from 250 to 750 nm with a Shimadzu (model UV-1203) spectrophotometer. The peak height at 334 nm (optical depth,estimated as the height in millimeters of peak maximum from spectropho-tometric scan of methanol extract) was considered an estimate of the con-centration of UVR-absorbing compounds (42). For the mixed mesocosmsexperiments, MAAs were analyzed by chromatography. A two-step extrac-tion procedure was used (43) whereby labile MAAs were extracted first andkept aside, and the filters were re-extracted for less labile MAAs, a methoddeveloped to optimize the extraction efficiency of the diverse MAAassemblages. MAAs were then separated by reverse-phase isocratic HPLC,injecting 50 lL aliquots in a Phenosphere C-8 column (Phenomenex, 250 34.6 mm, 5 lm) protected by a Brownlee Spheri-5 RP-8 guard (AppliedBiosystems, 30 3 4.6 mm, 5 lm) in a mobile phase of 0.1% acetic acid in25% aqueous methanol (44). The MAAs were tentatively identified bycomparison of retention times and order of appearance with publishedresults, and from the absorption spectra of the various peaks. Peak areas andpublished extinction coefficients (45) were used to calculate the concen-tration of MAAs, which were then summed to give the concentration oftotal MAAs in order to compare with the spectrophotometric assay of thefixed-depth experiment.

Statistical analyses. Repeated measures analysis of variance (RMA-NOVA) were performed (Statistica, version 5.1) to determine the sig-nificance of the differences between treatments observed for the carbonassimilation rate, species abundance, total cell number, chl a and growthrate. Exposure days were the repeated factor. Normality was checked usinga one-sample Kolmogorov-Smirnov test, whereas the sphericity assumptionthat concerns variance homogeneity was checked using the Mauchley’stest. When significant differences were found, a post-hoc Tukey test was

performed to determine which factor differed. When interaction wassignificant or the assumptions of sphericity were not satisfied, a one-factoranalysis of variance (ANOVA) was performed (46).

The Levene’s test was used to check for homogeneity of variances (46)for temporal variability UVB, UVA and PAR irradiances.

RESULTS

Fixed-depth experiments

Light environment. PAR, UVA and UVB daily doses were variable

during the study period (max. 5 80 kJ/m2; min. 5 35 kJ/m2 for

UVB). However, there were no significant differences for temporal

variability in irradiances of any of these wavebands (F 5 0.89;

P . 0.05) (Fig. 2).

Phytoplankton biomass. Total cell number and chl a concentra-

tion showed a positive and significant correlation (Fig. 3A insert;

R 5 0.88, P , 0.001). Because a more complete data set is avail-

able for cell number than for chl a, biomass will hereafter be

expressed in cells mL�1.

Cell number increased from the beginning of the experiment,

when cells were exposed only to PAR irradiance (Fig. 3A). In

contrast, cell numbers decreased under UVR conditions on days 1

and 2 in both NUVB and WUVB treatments. After that, there was

an increase in abundance in both NUVB and WUVB treatments

until the end of the experiment. The exposure of cells to UVA

radiation (WUVB treatment) reduced cell number compared with

Figure 2. Variation in the daily doses of (A) UVB (280–320 nm) andUVA (320–400 nm), and (B) PAR (400–700 nm) at the sampling site,Ushuaia. Fixed-depth experiments were carried out 11–17 February 1998(period denoted by the thick line). Light measurements are included fora time before the experiments.

Photochemistry and Photobiology, 2006, 82 925

numbers found after the equivalent PAR treatment (RMANOVA,

P , 0.01). No significant differences in abundance were found

between NUVB and WUVB treatments (P . 0.05) suggesting that

the effect is primarily due to UVA.

Phytoplankton assemblages. RMANOVA analyses for overall

diatom abundance (cell L�1) showed no differences between

treatments (Fig. 4A). Significantly higher diatom concentrations

were found on days 5 and 6 for all the treatments (RMANOVA,

Figure 3. Fixed-depth experiment: (A) Ln (natural logarithm) of total cellnumber (cell mL�1) (insert: Ln chl a as a function of exposure days). Eachpoint represents the mean; vertical lines are standard deviations. (B)Instantaneous growth rate using cell number when exposed to the naturalsolar radiation as a function of the exposure days. (C) Percentage ofinhibition induced by UVA during the exponential phase of growth withregard to PAR control.

Figure 4. Fixed-depth experiment: Abundance of diatoms and phyto-flagellates (A and B, respectively) when exposed to the different treatments:NUVB (UVR þ PAR); WUVB (UVA þ PAR) and PAR (PAR); (C)Relative abundance of phytoflagellates when exposed to the differenttreatments; (D) Variation in the relative abundance of phytoplankton taxa(phytoflagellates:diatoms ratio) as a function of time.

!


P , 0.001). There was a significant interaction between treatments

over time. Phytoflagellates showed a significant interaction be-

tween factors (treatments and days) and the daily analyses

showed a decrease in the WUVB treatment with regards to PAR

(RMANOVA, P , 0.001) between days 2 and 6 (Fig. 4B). No

differences in total (diatom þ phytoflagellates) cell number were

found between NUVB and WUVB for days 1, 5 and 6

(RMANOVA, P . 0.05).

Analyzed in relative terms (%), diatoms increased significantly

(P , 0.0001) with time in all treatments while phytoflagellates

decreased (Table 1, Fig. 4C). The RMANOVA for the relative

abundance of phytoplankton groups showed significant differences

between days of exposure (P , 0.0001; Fig. 4B). However, no

significant differences (P . 0.05) among treatments could be

found. The absence of interaction in the RMANOVA results did

not allow for daily ANOVA comparisons. Although it was not

significant, a higher percentage of diatoms occurred on day 6 in

the PAR treatment than in any of the UVR treatments (Fig. 4C).

The phytoflagellates:diatoms ratio showed no significant differ-

ences among treatments (RMANOVA, P . 0.05). However, there

was a decrease in phytoflagellate abundance during the first day of

exposure to UVR, suggesting inhibition of reproduction by UVB

and UVA, as shown by the differences observed between the

NUVB and WUVB treatments with respect to PAR (P , 0.05).

Inhibition was observed on day 2 under UVA conditions, whereas

on day 3 and until the end of experiment, no differences among

treatments were observed (Fig. 4D).

The most abundant diatom taxa found during the study are

presented in Table 1B. Chaetoceros sp. and Thalassiosira sp.

showed the highest (49% and 4%, respectively) increase under

UVB (NUVB treatment), compared with that under PAR þ UVA

(WUVB treatment) and PAR. Chaetoceros sp. also increased under

the WUVB treatment. An important increase in the abundance of

cells of the genus Skeletonema costatum was found at the end

of the experiment, although differences between treatments were

not significant. Small centric (;20 lm in diameter) and pennate

diatoms were more sensitive to UVB in terms of growth (in the

NUVB treatment) compared with growth under the PAR þ UVA

(WUVB) and PAR-only treatments. Small diatoms (5–20 lm in

diameter) were also affected by UVA, as shown by their low

abundance under this treatment in comparison with the others.

Photosynthesis and growth response to UVR. To determine the

degree of photosynthetic inhibition, assimilation numbers (lg C *

lg chl a�1 * h�1) of phytoplankton exposed to UVR (WUVB and

NUVB treatments) were compared with those exposed to PAR. At

the beginning (day 0) of the experiment, no UVR photosynthesis

inhibition was observed (Fig. 5). On day 2, when cloud conditions

increased the relative UVB exposure (Table 2), a 50% inhibition of

the photosynthetic rate was observed in the NUVB treatment; no

differences between the WUVB and PAR treatment were found

(Table 3, Fig. 5). On day 6 (the last day), no inhibition was ob-

served by UVR.

Instantaneous growth rate of the total phytoplankton community

was inhibited during day 1 by UVBþ UVAþ PAR (NUVB) and

UVA þ PAR (WUVB) radiation (180% and 140%, respectively)

compared with that under PAR (Table 4, Fig. 3B). Although cell

growth inhibition by UVA during the second day of exposure was

about 100%, no inhibition by UVB was observed. Growth rates in

WUVB decreased linearly at the end of the exponential phase

(day 3; Fig. 3C) when phytoflagellates continued dominating the

phytoplankton assemblage (Fig. 4C). After 6 days of exposure,

there was no UVR inhibition on growth. Chl a normalized MAAs

in NUVB-exposed cultures were significantly higher (P , 0.05)

than those corresponding to day 0 (Fig. 6).

Mesocosm experiments

Light environment. The daily fluence at the mesocosm surface

showed a high significant variability (F 5 2.55; P , 0.05) for the

various dates of the exposure due to the presence of clouds. UVB

doses presented maximum and minimum daily doses of 70 kJ/m2

and 30 kJ/m2, respectively. This variability was also evident in

PAR and UVA (Fig. 7). Mean depths of 1% light penetration for

PAR, UVA (340 nm) and UVB (305 nm) showed that these

wavelengths reached almost the bottom of the mesocosms (Table

5). The lamp-induced unweighted increase in UVR irradiance

(calculated from the five UVR wavelengths of the PUV in the

HUVB mesocosms relative to the NUVB treatment for a sunny day

Table 1. (A) Results of the RMANOVA, examining the effect of thetreatments and days on the relative abundances of diatoms along the fixed-depth experiment; (B) relative abundance (%) of most representative generain the community at the beginning and at the end of exposure.

A

Factor F P

Treatment* 1.82 . 0.05Day� 47.22 , 0.0001�Interaction 1.41 0.21

B

GenusInitial day

abundance (%)

Final day abundance (%)

NUVB WUVB PAR

Small centric diatoms 0.1 15 13 32Pennate diatoms 0.8 9 29 13S. costatum 0.1 3 7.5 3Thalassiosira sp. 0.8 4 0.5 0.8Chaetoceros sp. 0.1 49 29 8

*Treatments were NUVB (exposed to UVR þ PAR), WUVB (exposed toUVA þ PAR) and PAR. �Time of exposure to the different treatments.�Significant difference.

Figure 5. Fixed-depth experiment: Photosynthetic efficiency of phyto-plankton communities from the Beagle Channel as a function of radiation.Mean and standard deviation of means are presented. Significantdifferences between treatments are indicated with an asterisk.


at noon) was 1.72 times greater than natural irradiance. When

weighted with several biological weighting functions (BWFs) for

inhibition of photosynthesis, this relative increase was greater by

3.67-fold to 4.16-fold (47). These values are slightly high but in the

same range as some of the previous studies in mesocosms (48).

Phytoplankton assemblages. Microscopic results identified three

major groups: diatoms, small phytoflagellates and the eugleno-

phyte Eutreptiella sp. These groups correspond relatively well with

the pigment chemotaxonomy presented by Roy et al. (this issue).

The increase in diatom abundance coincided with the increase in

concentration of fucoxanthin (Roy et al., this issue). The highest

increase was observed in the abundance of ,3 lm phytoflagellates

and in the euglenophyte Eutreptiella sp. (11 lm wide 3 55 lm

long) in both HUVB and NUVB treatments (Fig. 8B,C). This

augmentation was higher in the HUVB treatment (Fig. 8B) than in

the NUVB treatment (Fig. 8C). In the latter treatment, the relative

abundance of Eutreptiella sp. was higher than in the other treat-

ments from day 5 to day 10.

Maximum phytoplankton abundance (as chl a concentration)

was observed on days 4–6, decreasing thereafter. No significant

differences between treatments were found (RMANOVA, P .

0.05; Fig. 9A).

The ratio of total chl a:chl a .5 lm showed an increase of algae

.5 lm between days 1 and 3 in all three treatments. In addition,

between days 3 and 5 the abundance of large-size (,5 lm) algae

increased in the HUVB treatment compared with abundances

after NUVB and WUVB treatments (Fig. 9B). On day 7, the ratio

increased without significant differences between treatments.

Because total chl a decreased on this day, the increase in the ratio

can be associated with a concomitant decrease in phytoflagellates

(Fig. 8).

Photosynthesis and growth response to UVR. No significant

differences between treatments (P . 0.05) were found in the in-

stantaneous growth rate of the phytoplankton community (calcu-

lated from chl a values) (RMANOVA, P . 0.05; Fig. 8A).

To determine photosynthetic inhibition by UVB, assimilation

numbers (lg C * lg chl a�1 * h�1) of phytoplankton exposed to

UVB (HUVB and NUVB treatments) were compared with phyto-

plankton exposed to WUVB treatment (Table 6). On day 1, ex-

posure to NUVB produced a 51% inhibition in photosynthesis with

regards to the WUVB treatment (Fig. 10). Higher assimilation

Table 3. (A) Results of the RMANOVA where the effect of thetreatments (NUVB, WUVB and PAR; see text) and time on algal carbonassimilation is evaluated during the fixed-depth experiment; (B) wheninteraction was significant (P , 0.05) each factor was analyzed by a one-factor ANOVA.

A

Factor F P

Treatment 10.59 , 0.01*Day 52.1 , 0.0001*Interaction 7.6 , 0.001*Day 0 1.57 . 0.05Day 2 8.59 , 0.01*Day 6 2.19 . 0.05

B

Average Homogeneity group�

Day 2, NUVB 0.25 ADay 2, WUVB 0.31 ADay 2, PAR 0.38 B

*Significant difference. �The same letter indicates that no significant dif-ferences (P . 0.05) were found (Tukey test).

Table 2. Experimental doses received by the phytoplankton assemblagesduring the days corresponding to the productivity studies during thefixed-depth experiment.

Exposure day UVB (kJ/m2) UVA (kJ/m2) PAR (kJ/m2)

0 29 404 35462 37 417 53206 28 447 4322

Table 4. (A) Results of the RMANOVA examining the effect of thetreatments and days on the instantaneous growth rate of the totalcommunity along the fixed-depth experiment; (B) when the interactionwas significant (P , 0.05), each factor was analyzed by a one-factorANOVA.

A

Factor F P

Treatment* 30.37 , 0.01�Day� 61.82 , 0.001�Interaction 3.03 , 0.05�Day 1 35.1 , 0.001�Day 2 12.24 , 0.01�Day 3 0.15 . 0.05

B

Average Homogeneity group§

Day 1, NUVB �0.63 ADay 1, WUVB �0.27 BDay 1, PAR 0.18 CDay 2, NUVB �0.03 ADay 2, WUVB �0.01 ADay 2, PAR 0.44 B

*Treatments were NUVB (exposed to UVR þ PAR), WUVB (exposed toUVA þ PAR) and PAR. �Time of exposure to the different treatments.�Significant difference. §The same letter indicates that no significant dif-ferences (P . 0.05) were found (Tukey test).

Figure 6. Fixed-depth experiment: Concentration of photoprotectiveMAAs measured at 334 nm optical depth (estimated as the height in mmof peak maximum from the spectrophotometric scan of a methanol extract)at the beginning and the end of the experiment, for the different radiationtreatments, normalized to total chl a. Mean and standard deviation of meansare presented.


numbers were evident on day 5 in the HUVB treatment. On day 6,

only NUVB samples were inhibited. At this time, the NUVB

samples were suddenly exposed to higher PAR and UVR levels

due to less cloudiness (Table 7). As in the previous fixed-depth

experiments, no differences were observed between treatments on

the last incubation days.

The chl a normalized total concentration of MAAs did not show

significant differences between treatments. The two treatments

exposed to UVB (NUVB and HUVB), showed higher values on

day 3. However, a decrease was observed between days 3 and 5,

after which concentrations remained low (Fig. 11). In contrast, the

photoprotective pigments of the xanthophyll cycle, diadinoxanthin

and diatoxanthin, showed an increase in their chlorophyll-specific

concentration with time (Fig. 12), but no significant differences

between treatments was found.

DISCUSSION

Under both fixed and mixed conditions, a strong inhibition in

photosynthesis was observed at the beginning of the experiment.

This inhibition was no longer observed by the end of the experi-

ments (Table 8). Reasons for this change differ among experi-

ments. Moreover, phytoplankton composition changed during the

experiment, and the change in the dominant microalgae group in

each case contributes to explain the differential adaptation to UVB

observed. In the fixed experiments, the initial phytoflagellate-

dominated community changed toward one dominated by dia-

toms. In the mixed experiments, both small phytoflagellates and

the euglenophyte Eutreptiella sp. were present throughout the

experiment. Diatoms synthesize MAAs as a response to UVR;

Eutreptiella has the capacity to produce changes in the xanthophyll

pigments, and thereby defend itself from UVB damage (Table 8).

Changes in the community structure and physiologicaladaptations in response to UVR

Several days of exposure to high UVB may induce changes in the

dominant species, from less to more tolerant ones (49,50). The

colony-forming Thalassiosira sp. grew differentially at the end of

the fixed-depth experiment. This genus has been well studied in

Antarctic environments, where it has been shown that it is capable

of fast synthesis of photoprotective compounds (21). Other centric

diatoms were, however, more sensitive to UVB, especially the

smaller (,30 lm) ones. Small cells—with a relatively high surface

to volume ratio—are more resistant to photosynthesis inhibition but

more vulnerable to DNA damage (51–53). The balance between

these two processes will determine their abundance under both

UVA and UVB exposure. Genera such as Chaetoceros, changing

from 0.1% to 49% under NUVB, and only to 8% under PAR, show

a great capacity to adapt to these radiations, probably due to

morphological changes (54) and to a great DNA repair potential

(i.e. in 6 h).

A progressive decrease in the ratio of phytoflagellates to diatoms

under UVB treatment was observed in the fixed-depth experiments.

In unialgal cultures, Hernando and San Roman (35) showed that

Figure 7. Variation in daily doses of (A) UVB (280–320 nm) and UVA(320–400 nm), and (B) PAR (400–700 nm) at the sampling site, Ushuaia.Well-mixed experiments were carried out 5–15 November 2001 (denotedby black line). Light measurements are included for a time before theexperiments.

Table 5. PAR, UVA (340 nm) and UVB (305 nm) conditions for surfaceincubations.

Variable

Experimental day

9 November(day 4)

12 November(day 7)

Daily fluence at surface*

PAR (kJ/m2) 50.59 47.47UVA (340 nm) (kJ/m2) 5.8 5.6NUV-B (305 nm) (kJ/m2) 0.3 0.3HUV-B (305 nm) (kJ/m2) 2.4 2.5

Daily fluence averaged over the water column

PAR (kJ/m2) 26.72 27.60UVA (340 nm) (kJ/m2) 1.7 1.7NUV-B (305 nm) (kJ/m2) 0.06 0.06HUV-B (305 nm) (kJ/m2) 0.40 0.39

Kd (m�1)�PAR 0.6 0.5UVA (340 nm) 1.4 1.3UVB (305 nm) 1.9 1.8

Z1% (m)�PAR 7.7 9.2UVA (340 nm) 3.3 3.5UVB (305 nm) 2.4 2.6

*Daily fluence at surface was corrected for the lamp shading effect. Thevalues presented are not wavelength-integrated and correspond to eachwavelength indicated. �Kd is light attenuation coefficient. �Z1% is depthof the euphotic zone.


sub-Antarctic phytoflagellates are extremely sensitive to UVB, in

contrast to diatoms of the genus Navicula. Therefore, in addition to

a higher tolerance of diatoms to UVR, phytoflagellate diminution

left an open niche for diatoms to occupy.

MAA concentrations in the fixed-depth incubation experiments

were higher under NUVB than in the other treatments, coinciding

with an increase in diatom abundance (i.e. day 6). The same trend

was observed in the mixed mesocosms, where the HUVB treatment

showed a maximum in the MAA to chl a ratio for day 7 (Fig. 11),

coinciding with a relative decrease in phytoflagellate and diatom

abundance (Fig. 9B), whereas euglenophytes (Eutreptiella sp.)

increased.

Effects of UVR exposure on phytoplankton fromsurface waters

Fixed-depth experiments simulate the environmental conditions to

which microalgae are exposed in stratified surface waters. The

relative contribution of UVA and UVB to the instantaneous growth

rate of the community varied throughout the time of exposure.

UVA was responsible for most of the observed inhibition in the

fixed-depth experiment, except during the first day when the

inhibition was produced by UVB. In addition, we found that such

UVA inhibition decreased with time, suggesting that cells had

acclimated to the new radiation conditions. UVA inhibition of

growth can be observed in the fixed-depth experiment during day 2

as shown for some other regions (55–57). This can be due to

a larger amount of UVA reaching the Earth even though UVB

causes more damage per unit energy (58). Cell number decreased

in the WUVB treatments while it increased in the PAR treatments,

although this inhibition ceased on day 3, at the end of the

exponential growth phase. Then, the UVB increase due to the

reduced cloud cover caused damage to phytoflagellates, allowing

the better adapted diatoms (probably due to the synthesis of

MAAs) to proliferate. Since no photosynthesis inhibition (i.e.reduction in carbon incorporation) was observed during the first

exposure days, phytoflagellates of this community would seem to

be resistant to UVB. However, these organisms were susceptible to

DNA damage, as denoted by growth inhibition.

Effects of UVR exposure in mixed mesocosms

Mixed situations are also common in Patagonian coasts, where

a shallow pycnocline can be disrupted by high-intensity winds. In

Figure 8. Mixed-depth mesocosm experiment: (A) Instantaneous growthrate using chl a L�1 when exposed to the natural solar radiation as a functionof the exposure days. (B) Total cell number of centric diatoms, Eutreptiellasp. and phytoflagellates in Ln (natural logarithm) cell number mL�1 forHUVB treatment. (C) Total cell number of centric diatoms, Eutreptiella sp.and phytoflagellates in Ln cell number mL�1 for NUVB treatment. Eachpoint represents the mean and vertical bars are standard deviation.

Figure 9. Mixed-depth mesocosm experiment: (A) Relative abundance ofphytoplankton communities using chl a concentration by L�1 when exposedto the different treatments: NUVB (UVRþ PAR); WUVB (UVAþ PAR)and PAR (PAR); (B) Variation in the relative abundance (ratio of chl atotal:chl a .5 um) as a function of time.


contrast with the results obtained for fixed-depth incubations, the

relative abundance of diatoms and phytoflagellates in the mixed

mesocosms did not show significant differences among treatments,

although lower abundance in phytoflagellates can be observed

when comparing the initial and final values. However, an increase

in Eutreptiella sp. is evident in the HUVB treatment. The presence

of Eutreptiella sp. can additionally be traced by the pigment

siphonein (Roy et al. in this issue). This is a derivative of b-

carotene, which acts as a pigment antenna in the photosynthesis

process (23) and is an antioxidant, protecting cells against

oxidative damage initiated by singlet oxygen (59).

Furthermore, the persistence of phytoflagellates in mesocosms

has an explanation in that when cells are mixed below the UVR

damaging zone, they can reduce damage either by activating DNA

and photosynthesis repair, and induce various photoprotective

mechanisms. Active repair of DNA damage seems to be effective

in phytoplankton assemblages from Patagonia, as seen in studies

carried out by Buma et al. (51) and Helbling et al. (52). Helbling

et al. (60) have shown the size dependence of DNA damage in

phytoplankton from Patagonian coastal waters, with small cells

(,2 um) being more sensitive to UVR than large cells. This is in

agreement with other studies carried out by Boelen et al. (61) and

Karentz et al. (54), which also suggest that DNA damage is

associated with the size structure of the community. Helbling et al.(52) showed that cyclobutane pyrimidine dimers (CPDs), an index

of DNA damage, accumulated in the surface (0.5 m), whereas no

accumulation was observed at depths of 3 and 6 m, in accordance

with the strong attenuation of DNA-effective UVR. The presence

Table 6. (A) Results of the RMANOVA where the effect of thetreatments (NUVB, WUVB and PAR; see text) and time on algal carbonassimilation for the different days is evaluated during the mixed mesocosmsexperiments; (B and C) when interaction was significant (P , 0.05) eachfactor was analyzed by a one factor ANOVA.

A

Factor F P

Treatment 1.5 . 0.05Day 87.57 , 0.000001*Interaction 2.55 , 0.01*

B

Day 1 11.98 , 0.01*Day 2 0.16 . 0.05Day 3 0.08 . 0.05Day 5 7.66 , 0.01*Day 6 8.74 , 0.01*Day 7 3.49 . 0.05Day 8 3.5 . 0.05Day 9 0.46 . 0.05Day 10 0.64 . 0.05

C

Average Homogeneity group�

Day 1

WUVB 2.76 BNUVB 1.35 A

Day 5

WUVB 1.66 ACNUVB 1.96 ACHUVB 2.34 AB

Day 6WUVB 1.03 BNUVB 0.71 AHUVB 1.09 Bc

*Significant difference. �The same letter indicates that no significant dif-ferences (P . 0.05) were found (Tukey test).

Figure 10. Mixed-depth mesocosm experiment: Photosynthetic efficiencyof phytoplankton communities from the Beagle Channel as a function ofradiation. Mean and standard deviation of means are represented.Significant differences between treatments are indicated with an asterisk.HUVB treatment started on day 3.

Table 7. Experimental doses (0.2 m depth and 6 h integrated) thatphytoplankton assemblages received during the days corresponding to theproductivity studies in the well-mixed mesocosm experiment.

Exposure day UVB (kJ/m2) UVA (kJ/m2) PAR (kJ/m2)

1 19 444 41972 22 441 41443 31 660 67605 22 419 40786 45 843 93477 43 796 87308 33 659 68829 22 474 4690

10 26 508 4974

Figure 11. Mixed-depth mesocosm experiment: Temporal change in thechl a normalized total concentration of MAAs (lg MAAs * lg chl a�1).Mean and standard deviation of means are shown.


of increasing lipid antioxidant after 3 days of UVB fixed-depth

exposure has been described for sub-Antarctic phytoflagellates in

previous studies (23). This mechanism would, however, not be

enough to counteract UVB damage at the surface.

Although phytoflagellates were also affected by UVB in the

mixed mesocosms, they were less intensely affected than in the

fixed-depth experiments. This was clear on day 7, after a high

exposure to UVB on day 6 (sampling was always performed at

0900 h). UVB surface values reached a maximum of 70 kJ/m2 in

the NUVB treatment (Fig. 7), where ozone concentration varied

between 330 and 370 Dobson units (DU). As in the fixed-depth

experiment during day 4 (ozone concentration varied between 305

and 320 DU), when phytoflagellates decreased, it appears that

there is a threshold UVB value over which the photoprotective

mechanisms acquired by phytoflagellates are not sufficient, and

DNA damage becomes important (51,62). Furthermore, small

phytoflagellates do not produce photoprotective compounds (60).

This might be associated with the elevated energetic cost that such

a synthesis represents for a small organism (63).

Photosynthetic inhibition

The degree of photosynthetic inhibition appeared to be related to

the light history of phytoplankton in both the fixed and mixed ex-

periments and to the composition of the phytoplankton assemblage.

No difference among treatments was observed at the beginning of

the fixed-depth treatment, and this might be explained by the high

abundance of phytoflagellates; similar results have been found

previously for the Beagle Channel phytoplankton (Hernando, un-

published). Moreover, van Leeuwe et al. (64) showed that the

flagellate Pyramimonas sp. (Prasinophyceae) displayed diel

rhythms in photosynthetic efficiency that primarily serve to prevent

photoinhibition during maximum irradiance at noon (65,66). The

high activity of the photoprotective xanthophyll cycle found in this

group (conversion of violaxanthin into the heat-dissipating pigment

zeaxanthin) suggested active control of photosynthetic efficiency

(67) under PAR irradiance.

During day 2 of the fixed-depth experiment, with higher UVR

doses (fewer clouds) than the previous days, photosynthetic rates

were inhibited by UVB, probably due to the accumulation of

effects after 2 days of exposure to an average radiation of 55 kJ/m2.

DNA, however, seems to be less affected, because no inhibition of

growth was observed. UVB can affect targets in the cell (DNA,

PSII) differentially, as suggested by Buma et al. (51) and Helbling

et al. (52) for temperate aquatic ecosystems. PSII is one of the

primary targets likely to be affected by UVR (68,69), and several

sites might be susceptible to damage (70–72), in association with

the D1 protein, which connects the primary electron donors with

the active PSII receptors. Contrasting with the results obtained in

the fixed-depth experiment, 50% inhibition of photosynthesis by

UVB was found on day 1 in the well-mixed mesocosm experiment.

Inhibition was higher than expected in a community also domi-

nated (.60%) by phytoflagellates (51–53). The inhibition might be

explained by the high UVB doses the community received the day

before (70 kJ/m2 at surface), due to little cloud cover, after having

been exposed to much lower irradiance levels during the preceding

days (i.e. around 45 kJ/m2). The change in irradiance conditions

probably affected the photosynthetic machinery of phytoflagellates

and diatoms with low MAA concentrations. Diatoms may be

affected more than phytoflagellates. Some (Antarctic) diatoms

possess limited means of rapid photoacclimation, due to a lack of

heterogeneity and flexibility of their photosynthetic membranes

compared with that of green algae (64,73,74). On day 5 of the well-

mixed mesocosm experiment, a 30% increase in carbon assimila-

tion was observed for algae exposed to HUVB compared with that

in the NUVB and WUVB treatments. Higher carbon assimilation

numbers were associated with the increased abundance of the

euglenophyte Eutreptiella sp., which was higher in the HUVB

treatment than in the NUVB treatment (25% and 15%, respec-

tively). The large size of this algal species results in a larger

influence on the biomass-related pigments. Results of experiments

by Roy et al. (this issue) show higher levels of chl b and the

photoprotectant siphonein on day 5 in the HUVB treatment

compared with levels in the NUVB and WUVB treatments. We

believe these larger assimilation numbers under the HUVB

treatments are caused by a reduction in photoinhibition resulting

from the change in community structure and increased photo-

protection. This may lead to a more efficient utilization of PAR

when cells are photoprotected, and also to the possible utilization

of UVA as an additional energy source for photosynthesis (75). In

addition, it has been noted that in the mesocosm experiment the D1

protein degradation rate gradually slowed down under the NUVB

and HUVB treatments (30). The gradually slower D1 degradation

may be partly related to the increase in other photoprotection

mechanisms, such as the xanthophyll cycle pigments (Fig. 12) or to

Figure 12. Mixed-depth mesocosm experiment: Temporal change in thechl a normalized concentration of diadinoxanthin and diatoxanthin (wt:wt).Means and standard deviation of means are shown.

Table 8. Summary of UVB effect on phytoplankton from the Beagle Channel exposed to solar and enhanced irradiance with and without mixing.

Mixing conditions UVB treatment Phytoplankton group* Photosynthetic first days Inhibition last day Interpretation

Fixed þUVB Diatoms 50% 0% MAA synthesisFixed �UVB Flagellates 0% 0%Mixed þUVB Eutreptiella sp. 51% 0% Diadinoxanthin and

diatoxanthin synthesisMixed �UVB Eutreptiella sp. and flagellates 0% 0%

*Denotes the taxa that showed differential growth under the experimental conditions.


the presence of siphonein, as mentioned above. The 30% inhibition

observed on day 6 in the NUVB treatment compared with that of

the other two treatments coincided with the highest UVB value

measured (45 kJ/m2, twice as much as in the previous day) due to

a reduced cloud cover.

By day 7, Eutreptiella sp. abundance doubled and phytoflagel-

lates decreased. Eutreptiella sp., already protected from UVR,

continued growing and showed no photosynthetic inhibition under

a high UVR dose, similar to that of day 6. Because the xanthophyll

cycle of euglenophytes uses diadinoxanthin and diatoxanthin, in

contrast to other chl b–containing algae (76), the high level of these

photoprotective pigments in the HUVB treatment (Fig. 12) con-

firms the role of photoprotection.

CONCLUSIONS

Fixed and mixed conditions represent two possible scenarios in

coastal sub-Antarctic waters seasonally exposed to the ozone hole,

where coastal waters can be both stratified or mixed, and therefore

present different UVR environments for phytoplankton. As shown

in Tables 3, 4 and 6, local microalgae can acclimate to high

irradiance conditions, for both growth rate and photosynthesis, in

agreement with other studies from this region (18,77). The struc-

ture of the phytoplankton community seems to play a central role

in this response, both under fixed-depth and mixed conditions.

Phytoflagellates, diatoms and other groups can grow differentially

in either scenario, developing different (group-specific) defense

and protection mechanisms. Furthermore, the contrast between

fixed-depth and mixed experiments showed that species can cope

with UVR. Under fixed light, MAA synthesis seems the most

effective photoprotective mechanism. Under variable light con-

ditions, Eutreptiella sp. was successfully protected by the synthesis

of diadinoxanthin and diatoxanthin.

Acknowledgements—This research was supported by a team grant fromthe Inter American Institute for Global Change (M. Vernet, lead principalinvestigator), by grants from the Natural Sciences and Engineering Coun-cil of Canada (NSERC) to Demers et al. (Special Research Opportunity)and partially by student stipends from CONICET, Argentina. The scintil-lation counter was provided by Universidad de Buenos Aires. We thankthe technical teams, Oscar Gonzales for his help with the incubations inmesocosms and Miguel Barbagallo for technical assistance with data col-lection during fixed-depth experiments and Leonardo Cantoni for lightdata processing. We especially thank Dr. San Roman for the use of hislaboratory installations and equipment.

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